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Frequently Asked Questions | Virus Production

flashBAC™ Baculovirus Expression Systems

flashBAC™ Baculovirus Expression Systems are a streamlined platform for the production of recombinant baculoviruses. Genetic optimization of the Autographa californica nucleopolyhedrovirus (AcMNPV) genome yields recombinant virus in a single step and eliminates the need for separation of recombinant and parental virus by plaque purification. flashBAC™ systems and pOET Insect and BacMam Transfer Plasmids are optimized for use with TransIT®-Insect Transfection Reagent.

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in the flashBAC™ Baculovirus Expression System?
Q2. How does the flashBAC™ Baculovirus Expression System work?
Q3. Which of the three flashBAC™ Baculovirus Expression Systems should I choose for expressing my gene of interest?
Q4. Can the pAcRP23-lacZ positive control vector be used as a cloning vector?
Q5. What is the size of the flashBAC™ DNA?
Q6. Which transfer vectors are compatible with flashBAC™ Expression System DNA?
Q7. What is a BacMam vector? What cells can I use with the BacMam virus?
Q8. What transfection reagent is recommended for use with flashBAC™ Baculovirus Expression System?
Q9. What is the mechanism of action of TransIT®-Insect Transfection Reagent?
Q10. Which insect cell lines are suitable for the flashBAC™ Baculovirus Expression System?
Q11. Do I need to perform a plaque assay before I begin protein production experiments?
Q12. How do the flashBAC™ Baculovirus Expression Systems compare to other commercially available baculovirus expression systems?
Q13. What titers can I expect using the flashBAC™ Expression System?
Q14. How should the flashBAC™ Expression System components be stored?

PROTOCOL QUESTIONS AND ANSWERS

Q15. Which serum-free medium should I use for preparing flashBAC™ DNA + transfer vector DNA + TransIT®-Insect transfection complexes?
Q16. Will antibiotics interfere with my transfection?
Q17. Is a media change required post-transfection?
Q18. What incubation conditions should be used for baculovirus production?
Q19. How can I validate delivery of the flashBAC™ Baculovirus Expression System?
Q20. How long can I store my baculovirus stocks?

TROUBLESHOOTING QUESTIONS

Q21. What can I do to further improve my baculovirus amplification titers with the flashBAC™ Baculovirus Expression System?

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in the flashBAC™ Baculovirus Expression Systems?
All flashBAC™ Baculovirus Expression Systems include flashBAC™ DNA and the pAcRP23-lacZ positive control plasmid. They are provided in either 5 or 24 reaction quantities.

Q2. How does the flashBAC™ Baculovirus Expression System work?
To generate recombinant baculovirus using the flashBAC™ System, insect cells are transfected with TransIT® -Insect Transfection Reagent, flashBAC™ DNA, and a transfer plasmid (e.g. pOET1, pOET1C_6xHIS, or pOET6) containing the gene of interest. Homologous recombination within insect cells inserts the gene of interest under the control of the polyhedrin (polh) promoter, and restores the function of ORF1629 allowing viral DNA to replicate and produce virus particles. The recombinant virus genome replicates to produce baculovirus that can be harvested directly from the culture medium of transfected insect cells.

Q3. Which of the three flashBAC™ Baculovirus Expression Systems should I choose for expressing my gene of interest?
The right flashBAC™ System for expressing your gene of interest will depend on the characteristics of the protein being expressed and the overall goal of the experiment. For general guidelines, please refer to the following:

  • flashBAC™: With the original flashBAC™ expression vector, protein expression and secretion is improved by deletion of the chitinase gene from the Autographa californica nucleopolyhedrovirus (AcMNPV) genome. This vector is suitable for expression of most simple nuclear or cytoplasmic recombinant proteins.
  • flashBAC™ GOLD: flashBAC™ GOLD builds upon the basic flashBAC™ technology with the deletion of the chitinase and cathepsin protease genes from the AcMNPV genome. This system is ideal for high level expression of membrane and secreted proteins as well as proteins that are more susceptible to degradation.
  • flashBAC™ ULTRA: flashBAC™ ULTRA is the most advanced flashBAC™ vector system and is optimized by deletions in the chitinase, cathepsin, p10, p74 and p26 genes. flashBAC™ ULTRA is the ideal system for the most difficult to express recombinant proteins including nuclear, cytoplasmic, membrane and secreted proteins.

Q4. Can the pAcRP23-lacZ positive control vector be used as a cloning vector?
The pAcRP23-lacZ contains a lacZ gene under control of the polyhedrin (polh) promoter and does not contain a multiple cloning site. The control vector is meant to confirm recombination within insect cells and is not recommended for subcloning.

Q5. What is the size of the flashBAC™ DNA?
The flashBAC™ Baculovirus Expression System is based on a modified AcMNPV genome and is approximately 136 kb in size.

Q6. Which transfer vectors are compatible with flashBAC™ Expression System DNA?
The flashBAC™ system is compatible with all baculovirus transfer vectors that are based on homologous recombination in insect cells at the polyhedrin gene locus. Compatible transfer plasmids available through Mirus Bio LLC are as follows:

  • pOET1 (MIR 6150) and pOET1C_6xHis (MIR 6151) for high titer baculovirus production
  • pOET6 BacMam (MIR 6152) for high titer BacMam virus production

Other compatible examples include pBacPAK8/9, pAcUW31 and pBacPAK-His1/2/3 (BD Biosciences Clontech). pFastBac vectors, which are designed for site-specific transposition in E. coli using the Bac-to-Bac system (Life Technologies) are NOT compatible. A list of compatible plasmids sold through external sources can be found here (PDF).

Q7. What is a BacMam vector? What cells can I use with the BacMam virus?
A BacMam vector is a baculovirus transfer vector designed to facilitate the expression of foreign genes in mammalian cells under the cytomegalovirus (CMV) immediate early gene promoter. The purified baculovirus generated with insect cells can be used to transduce most mammalian cell types in a titer-dependent manner.

Q8. What transfection reagent is recommended for use with flashBAC™ Baculovirus Expression System?
We recommend TransIT®-Insect Transfection Reagent with the flashBAC™ Expression Systems. TransIT®-Insect Transfection Reagent is a proprietary, non-liposomal, animal-origin-free formulation developed for high transfection performance in insect cell lines.

Q9. What is the mechanism of action of TransIT®-Insect Transfection Reagent?
The TransIT®-Insect Reagent complexes with negatively charged DNA to form net positive lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are taken into the cell via endocytosis.

Q10. Which insect cell lines are suitable for the flashBAC™ Baculovirus Expression System?
Sf9 or Sf21 cells are generally used for co-transfections, virus amplification and plaque assays. T. ni cells are often used to maximize protein yields but are not recommended for virus production and amplification.

Q11. Do I need to perform a plaque assay before I begin protein production experiments?
Plaque purification to separate parental from recombinant virus is not necessary with the flashBAC™ Expression System. For optimal protein yield, it is recommended that baculovirus titers be determined via plaque assay or a comparable method prior to infection of insect cells.

Q12. How do the flashBAC™ Baculovirus Expression Systems compare to other commercially available baculovirus expression systems?
The flashBAC™ Expression System is the simplest and least time-consuming baculovirus expression system available that yields 100% recombinant virus in a single step. For direct competitor comparisons, please see the flashBAC™ Baculovirus Expression System product page.

Q13. What titers can I expect using the flashBAC™ Expression System?
Baculovirus titers will vary based on the properties of the gene of interest cloned into the transfer vector. A titer of approximately 1 x 107 pfu/ml at day 5 post-transfection is expected with the pAcRP23-lacZ control plasmid, and titers of ≥ 1 x 108 are typically observed after P1 amplification.

Q14. How should the flashBAC™ Expression System components be stored?
Store the components as follows:

  • Store flashBAC™ DNA at 4°C. DO NOT FREEZE.
  • Store Control Transfer Plasmid (pAcRP23-lacz) at -20°C.

PROTOCOL QUESTIONS AND ANSWERS

Q15. Which serum-free medium should I use for preparing flashBAC™ DNA + transfer vector DNA + TransIT®-Insect transfection complexes?
Always prepare TransIT®-Insect Reagent:DNA complexes in serum-free growth medium. Mirus recommends Grace’s Insect Basal Medium.

Q16. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q17. Is a media change required post-transfection?
No, a media change is not required following transfection. However, media will need to be added back into transfected wells 5-24 hours post-transfection. For further details, see section I-D of the full protocol (PDF).

Q18. What incubation conditions should be used for baculovirus production?
The optimal temperature for Sf9 and Sf21 growth and infection is 27-29°C. If the insect culture medium used employs a phosphate buffering system (e.g. Grace’s, SF-900), a CO2 incubator is not required. Use vented shake, spinner, or tissue culture flask caps to ensure adequate dissolved oxygen content.

Q19. How can I validate delivery of the flashBAC™ Baculovirus Expression System?
To verify efficient transfection, use TransIT®-Insect Transfection Reagent to deliver the positive control plasmid provided in the flashBAC™ kit (pAcRP23-lacZ). LacZ-positive virus plaques can be stained by following the plaque assay procedure outlined in Section III of the flashBAC™ Baculovirus Expression Systems full protocol (PDF) using X-gal rather than Neutral Red.

Q20. How long can I store my baculovirus stocks?
Baculovirus stocks may be stored for up to 12 months at 4°C, though loss of titer can occur during this time. To minimize titer loss, add 2-5% serum. If the baculovirus stock is stored for greater than 3 months, titer the virus before use and re-amplify if necessary. For long-term storage, virus should be aliquoted and stored at –80°C. Due to reduction of virus titer by freezing, avoid multiple freeze thaws and re-amplify virus before use. Storage at –20°C or in liquid nitrogen is not recommended.

TROUBLESHOOTING QUESTIONS

Q21. What can I do to further improve my baculovirus amplification titers with the flashBAC™ Baculovirus Expression System?
Multiple factors can contribute to transfection efficiency and ultimate baculovirus titer; refer to the following critical troubleshooting tips to improve your transfection results:

  • Cell density (% confluence) not optimal at time of transfection - See the full protocol (PDF) for recommended plating and amplification recommendations. Baculovirus replication is inhibited when cells are seeded too densely. Depending on the cell type, higher or lower densities may be required for optimal amplification.
  • Cells not actively dividing at the time of transfection - Maintain insect cells used for the production of recombinant viruses in log-phase growth and discard after passage 30. Subculture cells before they become overgrown and enter stationary phase, generally every 3-4 days. Cells used for transfection and amplification should be in log-phase growth with a viability of >95%.
  • Suboptimal P0 volume used for amplification - The virus titer from the co-transfection may be too low for the volume of cells being infected. To determine the optimal harvest time, leave the infection for longer than 5 days and monitor virus amplification using a phase-contrast microscope. Conversely, viral titers will be lower if too much virus was added to the culture of cells and only one round of amplification occurred.
  • Poor quality of P0 virus stock - If the P0 stock has been stored for longer than 3 months, you may need to re-titer. If the titer has decreased, increase the volume of P0 used for amplification.
  • Culture time post-transfection - Determine the optimal transfection incubation time for each cell type and experiment. Test a range of incubation times. For P0 and P1 baculovirus production, the best incubation time is generally 4-5 days.

For additional tips, please refer to the Troubleshooting Guide in the user protocol (PDF).

TransIT® Lentivirus System

Lentivirus production platforms are often limited by insufficient viral titers which require concentration before use. The TransIT® Lentivirus System was developed for enhanced delivery of the essential transfer and packaging vectors required for higher-titer lentivirus production. With the TransIT® Lentivirus System, greater than 2-fold higher functional titers are achieved over competing lentivirus production systems.

GENERAL QUESTIONS AND ANSWERS

Q1. What is lentivirus?
Q2. How is lentivirus produced?
Q3. What components are included in the TransIT® Lentivirus System?
Q4. What is the composition of the TransIT®-Lenti Transfection Reagent?
Q5. What vectors/elements are included in the Lentivirus Packing Mix Powered by MISSION® Genomics?
Q6. What is the shelf life of the TransIT® Lentivirus System?
Q7. How should the TransIT® Lentivirus System components be stored?
Q8. Can the TransIT®-Lenti Transfection Reagent be used to generate lentivirus in both adherent and suspension cells?
Q9. What titers are expected when using the TransIT® Lentivirus System?
Q10. How does TransIT® Lentivirus System compare to other commercially available lentiviral packaging systems?

PROTOCOL QUESTIONS AND ANSWERS

Q11. Which serum-free medium should I use for preparing TransIT®-Lenti:DNA transfection complexes?
Q12. How much TransIT®-Lenti Reagent and total DNA (packaging + transfer plasmids) should be used for transfection?
Q13. What is the recommended transfer:packaging plasmid ratio for optimal lentivirus generation?
Q14. Will antibiotics interfere with my transfection?
Q15. Is a media change required post-transfection?
Q16. Are cell density and passage number important factors for successful transfection?
Q17. At what time point do you recommend harvesting lentivirus post-transfection?
Q18. How can I titer my lentivirus stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q19. What can I do to further improve my lentivirus titers when using TransIT®-Lenti?
Q20. Should I be concerned about cellular toxicity when using TransIT®-Lenti Transfection Reagent?

GENERAL QUESTIONS AND ANSWERS

Q1. What is lentivirus?
Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family commonly used as a gene delivery tool for robust and stable transgene expression in both dividing and non-dividing cells. Stable transgene expression is achieved through the random integration of the viral genome into the host cell genome.

Q2. How is lentivirus produced?
To produce lentivirus, cells are co-transfected with packaging plasmids encoding the required gagpol, and rev structural and regulatory genes and a transfer vector encoding the gene of interest (GOI). Essential components are combined with the vesicular stomatitis virus (VSV) envelope protein G for a broad virus tropism and increased stability during purification procedures. The lentivirus particles are secreted into the culture medium where they are collected, filtered and frozen into aliquots for subsequent transduction into target cells.

Q3. What components are included in the TransIT® Lentivirus System?
The TransIT® Lentivirus System contains TransIT®-Lenti Transfection Reagent and Lentiviral Packaging Mix Powered by MISSION® Genomics for high-titer lentivirus production in adherent HEK 293T cell types. TransduceIT™ Reagent is also included in the kit for enhanced lentiviral transduction of target cells.

Q4. What is the composition of the TransIT®-Lenti Transfection Reagent?
TransIT®-Lenti Transfection Reagent is a proprietary, animal origin-free formulation composed of a lipid and polymer mixture.

Q5. What vectors/elements are included in the Lentiviral Packaging Mix Powered by MISSION® Genomics?
The Lentiviral Packaging Mix Powered by MISSION® Genomics contains an optimized mixture of the following two plasmids:

  • pMISSION GAG POL - This packaging vector contains the gagpol and rev genes required to generate the virion structural proteins and packaging functions. The tat transactivator element is also included for enhanced transcription of the viral genome.
  • pMISSION VSV-G - The vesicular stomatitis virus (VSV) G-protein vector provides the heterologous envelope for broad virus tropism and increased stability during purification procedures.

These plasmids represent a hybrid second/third generation lentivirus packaging system and are compatible with both second and third generation lentivirus transfer plasmids.

Q6. What is the shelf life of the TransIT® Lentivirus System?
When properly stored and handled, the TransIT®-Lenti Transfection Reagent is guaranteed for 6 months from the date of purchase. The Lentiviral Packaging Mix Powered by MISSION® Genomics and TransduceIT™ Reagent are guaranteed for 1 year from the date of purchase.

Q7. How should the TransIT® Lentivirus System components be stored?
Store the TransIT®-Lenti Reagent, Lentiviral Packaging Mix Powered by MISSION® Genomics and TransduceIT™ Reagent at -20ºC. Before each use, warm to room temperature and vortex gently.

Q8. Can the TransIT®-Lenti Transfection Reagent be used to generate lentivirus in both adherent and suspension HEK 293 cells?
No. TransIT®-Lenti Transfection Reagent is only recommended for adherent cell transfections (e.g. HEK 293T/17) and is not recommended for lentivirus generation in suspension cell types. For highest lentivirus titers in suspension 293 cell types, we recommend using our TransIT-PRO® Transfection Reagent (PDF).

Q9. What titers are expected when generating lentivirus with the TransIT® Lentivirus System?
Lentivirus titers vary widely and ultimately depend on the HEK 293T cell subtype, packaging system and transfer vector used for lentivirus generation. Functional titers of ≥ 107 are typically achieved with the TransIT® Lentivirus System when using a MISSION® powered transfer plasmid in HEK 293T/17 cells. For more information, see the TransIT® Lentivirus System product page.

Q10. How does TransIT® Lentivirus System compare to other commercially available lentiviral packaging systems?
Greater than 2-fold higher functional titers are achieved over other commercially available lentiviral production systems when using TransIT® Lentivirus System with a MISSION® powered transfer plasmid. For comparison data, see the TransIT®-Lenti Expression System product page.

PROTOCOL QUESTIONS AND ANSWERS

Q11. Which serum-free medium should I use for preparing TransIT®-Lenti:DNA transfection complexes?
For best results, we recommend using Opti-MEM® I Reduced-Serum Medium for preparing TransIT®-Lenti:DNA complexes. Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes.

Q12. How much TransIT®-Lenti and total DNA (packaging + transfer plasmid) should be used for transfection?
The quantities of TransIT®-Lenti Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel (see Table 1 in the TransIT®-Lenti Full Protocol (PDF)). In each well of a 6-well plate, our starting recommendation is 6 µl TransIT®-Lenti Reagent and 2 µg total DNA (1 µg transfer plasmid + 1 µl Packaging Mix) which represents a 3:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q13. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation? 
The TransIT®-Lenti Reagent was optimized using a pre-mix of lentivirus packaging vectors. If using individual packaging plasmids, we recommend a starting ratio of 4 µg gag-pol vector, 1 µg rev vector and 1 µg VSV-G vector. Premix the packaging plasmids with an equal amount of the transfer vector (e.g. 6 µg) to maintain a 1:1 (wt:wt) ratio of packaging to transfer plasmids.

Q14. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q15. Is a media change required post-transfection?
No. A media change to remove TransIT®-Lenti:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus titers.

Q16. Are cell density and passage number important factors for successful transfection?
Yes. A cell density of ≥80% confluence at the time of transfection is essential for optimal transfection using TransIT®-Lenti Reagent in cell types typically used for lentivirus generation (e.g. HEK 293T/17). Transfecting at less than 80% confluence may lead to increased cytotoxicity and decreased lentivirus titers. Monitoring passage number is important since high passage numbers can alter cell characteristics such as morphology and division rate. Maintain HEK 293T/17 cells below passage 30 for optimal recombinant lentivirus production.

Q17. At what time point do you recommend harvesting lentivirus post-transfection?
The optimal incubation time for harvesting lentivirus is 48 hours post-transfection. Minimal amounts of functional lentivirus are produced during the period of 48-72 hours post-transfection.

Q18. How can I titer my lentivirus stock?
p24 ELISA assays designed to measure viral-associated p24 capsid protein and qPCR kits designed to amplify a conserved region of the HIV-1 genome (e.g. gag) are common methods for titering lentivirus. However, these methods measure the total amount of viral protein or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The TransIT®-Lenti Full Protocol (PDF) describes a method to determine functional titer by transducing HEK 293T/17 cells with a GFP reporter lentivirus. In this method, cells are tranduced at a low MOI and assayed for GFP expression after 48 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (Transducing Units/ml) = Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]                                                                                                                                                     (Volume of Lentivirus Stock in ml)

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q19. What can I do to further improve lentivirus titers when using TransIT®-Lenti?
Multiple factors can contribute to lentiviral titers; refer to the following critical troubleshooting tips to improve your transfection results:

  • Cell density (% confluence) not optimal at time of transfection - The recommended cell density for 293T cell types at the time of transfection with TransIT®-Lenti Reagent is ≥80% confluence.
  • Suboptimal TransIT®-Lenti Reagent to DNA ratio - We recommend using 3 µl TransIT®-Lenti Reagent per 1 µg total DNA (transfer + packaging plasmids).
  • Poor quality of DNA - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit .
  • Complexes added to cells in serum-free medium – Form complexes in serum-free medium (e.g. Opti-MEM® I Reduced-Serum Medium) and add to cells in complete (serum-containing) growth medium.
  • Insufficient quantities of DNA for transfection – If using an alternative packaging plasmid system, increasing the amount of total DNA (transfer + packaging plasmids) per reaction while maintaining the recommended 3:1 reagent-to-DNA ratio may help improve titers.

For additional tips, please refer to the Troubleshooting Guide (PDF) in the user protocol.

Q20. Should I be concerned about cellular toxicity when using TransIT®-Lenti Transfection Reagent?
Toxicity is not typically a concern when over-expressing VSV-G in cultures, as some morphology changes and cell to cell fusion events are expected and do not adversely affect lentivirus titers. However, cellular toxicity may be observed if the transfection protocol is not followed properly. The following are considerations for improving cell health post-transfection:

  • Cell density (% confluence) too low at time of transfection - The recommended cell density at the time of transfection is ≥80% confluence. If cytotoxicity is observed, increase the cell density to maintain cell health.
  • Plasmid DNA contains high level of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired, consider reducing the amount of target plasmid. Maintain the optimal TransIT®-Lenti Reagent:DNA ratio by using carrier DNA such as an empty cloning vector. For example, use 0.5 µg Transfer DNA, 0.5 µg pUC18 and 1 ug Packaging Plasmids for complexes with a reduced amount of target plasmid.
  • TransIT®-Lenti Reagent:DNA complexes were not mixed thoroughly with the cells in the well – Mix thoroughly to evenly distribute the complexes to all cells. Rock the dish back and forth and side to side to properly distribute the complexes. Do not swirl or rotate the dish, as this may result in uneven distribution.
  • Cell morphology has changed – If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
  • Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a freshly thawed stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide (PDF) in the user protocol.

TransIT®-Lenti Transfection Reagent

Lentivirus production platforms are often limited by insufficient viral titers which require concentration before use. The TransIT®-Lenti Transfection Reagent was developed for enhanced delivery of the essential transfer and packaging vectors required for higher-titer lentivirus production. With TransIT®-Lenti, greater than 2-fold higher functional titers are achieved over other commercially available transfection reagents commonly used for lentivirus generation.

GENERAL QUESTIONS AND ANSWERS

Q1. What is lentivirus?
Q2. How is lentivirus produced?
Q3. What is the composition of the TransIT®-Lenti Transfection Reagent?
Q4. What is the shelf life of the TransIT®-Lenti Transfection Reagent?
Q5. How should the TransIT®-Lenti Transfection Reagent be stored?
Q6. Can the TransIT®-Lenti Transfection Reagent be used to generate lentivirus in both adherent and suspension cells?
Q7. What titers are expected when using the TransIT®-Lenti Transfection Reagent?
Q8. How does TransIT®-Lenti compare to other commercially available transfection reagents?

PROTOCOL QUESTIONS AND ANSWERS

Q9. Which serum-free medium should I use for preparing TransIT®-Lenti:DNA transfection complexes?
Q10. How much TransIT®-Lenti Reagent and total DNA (packaging + transfer plasmids) should be used for transfection?
Q11. What is the recommended transfer:packaging plasmid ratio for optimal lentivirus generation?
Q12. Will antibiotics interfere with my transfection?
Q13. Is a media change required post-transfection?
Q14. Are cell density and passage number important factors for successful transfection?
Q15. At what time point do you recommend harvesting lentivirus post-transfection?
Q16. How can I titer my lentivirus stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q17. What can I do to further improve my lentivirus titers when using TransIT®-Lenti?
Q18. Should I be concerned about cellular toxicity when using TransIT®-Lenti Transfection Reagent?

GENERAL QUESTIONS AND ANSWERS

Q1. What is lentivirus?
Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family commonly used as a gene delivery tool for robust and stable transgene expression in both dividing and non-dividing cells. Stable transgene expression is achieved through the random integration of the viral genome into the host cell genome.

Q2. How is lentivirus produced?
To produce lentivirus, cells are co-transfected with packaging plasmids encoding the required gagpol, and rev structural and regulatory genes and a transfer vector encoding the gene of interest (GOI). Essential components are combined with the vesicular stomatitis virus (VSV) envelope protein G for a broad virus tropism and increased stability during purification procedures. The lentivirus particles are secreted into the culture medium where they are collected, filtered and frozen into aliquots for subsequent transduction into target cells.

Q3. What is the composition of the TransIT®-Lenti Transfection Reagent?
TransIT®-Lenti Transfection Reagent is a proprietary, animal origin-free formulation composed of a lipid and polymer mixture.

Q4. What is the shelf life of the TransIT®-Lenti Transfection Reagent?
When properly stored and handled, the TransIT®-Lenti Transfection Reagent is guaranteed for 1 year from the date of purchase.

Q5. How should the TransIT®-Lenti Transfection Reagent be stored?
Store the TransIT®-Lenti Reagent at -20ºC. Before each use, warm to room temperature and vortex gently.

Q6. Can the TransIT®-Lenti Transfection Reagent be used to generate lentivirus in both adherent and suspension HEK 293 cells?
No. TransIT®-Lenti Transfection Reagent is only recommended for adherent cell transfections (e.g. HEK 293T/17) and is not recommended for lentivirus generation in suspension cell types. For highest lentivirus titers in suspension 293 cell types, we recommend using our TransIT-PRO® Transfection Reagent (PDF).

Q7. What titers are expected when generating lentivirus with the TransIT®-Lenti Transfection Reagent?
Lentivirus titers vary widely and ultimately depend on the HEK 293T cell subtype, packaging system and transfer vector used for lentivirus generation. Functional titers of ≥ 107 are typically achieved with the TransIT®-Lenti Transfection Reagent when using a MISSION® powered Lentiviral Packaging Mix and transfer plasmid in HEK 293T/17 cells. For more information, see the TransIT®-Lenti Transfection Reagent.

Q8. How does TransIT®-Lenti compare to other commercially available transfection reagents?
Greater than 2-fold higher functional titers are achieved over other commercially available transfection reagents when using TransIT®-Lenti Transfection Reagent with a MISSION® powered transfer plasmid and Lentiviral Packaging Mix. For comparison data, see the TransIT®-Lenti Transfection Reagent.

PROTOCOL QUESTIONS AND ANSWERS

Q9. Which serum-free medium should I use for preparing TransIT®-Lenti:DNA transfection complexes?
For best results, we recommend using Opti-MEM® I Reduced-Serum Medium for preparing TransIT®-Lenti:DNA complexes. Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes.

Q10. How much TransIT®-Lenti and total DNA (packaging + transfer plasmid) should be used for transfection?
The quantities of TransIT®-Lenti Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel (see Table 1 in the TransIT®-Lenti Full Protocol (PDF)). In each well of a 6-well plate, our starting recommendation is 6 µl TransIT®-Lenti Reagent and 2 µg total DNA (1 µg transfer plasmid + 1 µl Packaging Mix) which represents a 3:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q11. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation? 
The TransIT®-Lenti Reagent was optimized using a pre-mix of lentivirus packaging vectors. If using individual packaging plasmids, we recommend a starting ratio of 4 µg gag-pol vector, 1 µg rev vector and 1 µg VSV-G vector. Premix the packaging plasmids with an equal amount of the transfer vector (e.g. 6 µg) to maintain a 1:1 (wt:wt) ratio of packaging to transfer plasmids.

Q12. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q13. Is a media change required post-transfection?
No. A media change to remove TransIT®-Lenti:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus titers.

Q14. Are cell density and passage number important factors for successful transfection?
Yes. A cell density of ≥80% confluence at the time of transfection is essential for optimal transfection using TransIT®-Lenti Reagent in cell types typically used for lentivirus generation (e.g. HEK 293T/17). Transfecting at less than 80% confluence may lead to increased cytotoxicity and decreased lentivirus titers. Monitoring passage number is important since high passage numbers can alter cell characteristics such as morphology and division rate. Maintain HEK 293T/17 cells below passage 30 for optimal recombinant lentivirus production.

Q15. At what time point do you recommend harvesting lentivirus post-transfection?
The optimal incubation time for harvesting lentivirus is 48 hours post-transfection. Minimal amounts of functional lentivirus are produced during the period of 48-72 hours post-transfection.

Q16. How can I titer my lentivirus stock?
p24 ELISA assays designed to measure viral-associated p24 capsid protein and qPCR kits designed to amplify a conserved region of the HIV-1 genome (e.g. gag) are common methods for titering lentivirus. However, these methods measure the total amount of viral protein or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The TransIT®-Lenti Full Protocol (PDF) describes a method to determine functional titer by transducing HEK 293T/17 cells with a GFP reporter lentivirus. In this method, cells are tranduced at a low MOI and assayed for GFP expression after 48 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (Transducing Units/ml) = Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]                                                                                                                   (Volume of Lentivirus Stock in ml)

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q17. What can I do to further improve lentivirus titers when using TransIT®-Lenti?
Multiple factors can contribute to lentiviral titers; refer to the following critical troubleshooting tips to improve your transfection results:

  • Cell density (% confluence) not optimal at time of transfection - The recommended cell density for 293T cell types at the time of transfection with TransIT®-Lenti Reagent is ≥80% confluence.
  • Suboptimal TransIT®-Lenti Reagent to DNA ratio - We recommend using 3 µl TransIT®-Lenti Reagent per 1 µg total DNA (transfer + packaging plasmids).
  • Poor quality of DNA - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit .
  • Complexes added to cells in serum-free medium – Form complexes in serum-free medium (e.g. Opti-MEM® I Reduced-Serum Medium) and add to cells in complete (serum-containing) growth medium.
  • Insufficient quantities of DNA for transfection – Increasing the amount of total DNA (transfer + packaging plasmids) per reaction while maintaining the recommended 3:1 reagent-to-DNA ratio may help improve titers.

For additional tips, please refer to the Troubleshooting Guide (PDF) in the user protocol.

Q18. Should I be concerned about cellular toxicity when using TransIT®-Lenti Transfection Reagent?
Toxicity is not typically a concern when over-expressing VSV-G in cultures, as some morphology changes and cell to cell fusion events are expected and do not adversely affect lentivirus titers. However, cellular toxicity may be observed if the transfection protocol is not followed properly. The following are considerations for improving cell health post-transfection:

  • Cell density (% confluence) too low at time of transfection - The recommended cell density at the time of transfection is ≥80% confluence. If cytotoxicity is observed, increase the cell density to maintain cell health.
  • Plasmid DNA contains high level of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired, consider reducing the amount of target plasmid. Maintain the optimal TransIT®-Lenti Reagent:DNA ratio by using carrier DNA such as an empty cloning vector. For example, use 0.5 µg transfer DNA, 0.5 µg pUC18 and 1 ug packaging plasmids for complexes with a reduced amount of target plasmid.
  • TransIT®-Lenti Reagent:DNA complexes were not mixed thoroughly with the cells in the well – Mix thoroughly to evenly distribute the complexes to all cells. Rock the dish back and forth and side to side to properly distribute the complexes. Do not swirl or rotate the dish, as this may result in uneven distribution.
  • Cell morphology has changed – If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
  • Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a freshly thawed stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide (PDF) in the user protocol.

TransIT-VirusGEN® Reagent

The TransIT-VirusGEN® Reagent enables high-titer lentivirus and AAV production in serum-containing adherent cultures, as well as serum-free suspension 293-derived cell types. The TransIT-VirusGEN® and TransIT-VirusGEN® GMP Transfection Reagents are identical in formulation, which equips researchers with the same reliable and scalable performance and streamlined viral vector production workflow as they progress from discovery and development to clinical trials and commercial manufacturing. Building on our commitment to product quality, the TransIT-VirusGEN® GMP Transfection Reagent is manufactured under Current Good Manufacturing Practices (cGMP), which are in compliance with the regulations defined in U.S. 21 CFR 820 from the FDA.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the TransIT-VirusGEN® Reagent?
Q2. What is the difference between TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
Q3. Is a functional AAV titer assay performed for quality release testing of TransIT-VirusGEN® GMP Transfection Reagent?
Q4. What additional analytical assays are available for the TransIT-VirusGEN® Reagent?
Q5. What configurations are available for TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
Q6. What is the shelf life of the TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
Q7. How should the TransIT-VirusGEN® Reagent be stored?
Q8. Can the TransIT-VirusGEN® Reagent be used to generate lentivirus and AAV in both adherent and suspension HEK 293 cells?
Q9. What titers are expected when generating virus with the TransIT-VirusGEN® Reagent?

PROTOCOL QUESTIONS AND ANSWERS

Q10. Which serum-free medium should I use for preparing TransIT-VirusGEN® Reagent:DNA transfection complexes?
Q11. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Q12. For lentivirus generation, how much TransIT-VirusGEN® Reagent and total DNA (packaging + transfer plasmid) should be used for transfection?
Q13. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation?
Q14. For AAV generation, how much TransIT-VirusGEN® Reagent and total DNA (GOI containing transfer + rep/cap + pHelper) should be used for transfection?
Q15. What is the recommended plasmid ratio for optimal AAV generation?
Q16. How should transfections with TransIT-VirusGEN® Reagent be scaled if using culture vessels not listed in the Full Protocol?
Q17. Will antibiotics interfere with my transfection?
Q18. Is a media change required post-transfection?
Q19. Are cell density and passage number important factors for successful transfection?
Q20. At what time point do you recommend harvesting lentivirus post-transfection?
Q21. How can I titer my lentivirus stock?
Q22. At what time point do you recommend harvesting AAV post-transfection?
Q23. How can I titer my AAV stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q24. How can I further improve titers when using TransIT-VirusGEN® Reagent?
Q25. Should I be concerned about cellular toxicity when using TransIT-VirusGEN® Reagent?

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent is a proprietary, animal origin-free formulation composed of a lipid and polymer mixture.

Q2. What is the difference between TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
Both products are the same formulation, but each product has different quality control release requirements:

  • The TransIT-VirusGEN® Transfection Reagent is a research-grade product which is released based on appearance and a functional AAV titer assay for all volume configurations. Additional release testing (sterility, endotoxin, and mycoplasma) is performed for configurations that are 1.5 ml or greater in volume.
  • The TransIT-VirusGEN® GMP Transfection Reagent is a GMP-grade product which is manufactured in accordance with the Mirus Bio Quality Management System, which incorporates the appropriate cGMP product and production requirements. The product is manufactured in audited facilities that are certified to ISO 13485 and comply with 21 CFR 820. Release testing for TransIT-VirusGEN® GMP Transfection Reagent consists of appearance, sterility, identity, mycoplasma and endotoxin assays.

Q3. Is a functional AAV titer assay performed for quality release testing of TransIT-VirusGEN® GMP Transfection Reagent?
A functional AAV titer assay is not performed for quality release testing of the TransIT-VirusGEN® GMP Transfection Reagent. The TransIT-VirusGEN® GMP Reagent is formulated through a validated manufacturing process which was established to ensure consistent functional AAV titer thereby removing the need to perform a functional AAV titer assay on each lot.

Q4. What additional analytical assays are available for the TransIT-VirusGEN® Reagent?
Residual reagent and identity tests are available for VirusGEN® products. Please contact techsupport@mirusbio.com for more information.

Q5. What configurations are available for TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
TransIT-VirusGEN® Transfection Reagent is available in 0.3 ml (MIR 6703), 0.75 ml (MIR 6704), 1.5 ml (MIR 6700), 5 × 1.5 ml (MIR 6705), 10 × 1.5 ml (MIR 6706), 30 ml (MIR 6720) and 150 ml (MIR 6740). TransIT-VirusGEN® GMP Transfection Reagent is available in 150 ml (MIR 6845-GMP).

Q6. What is the shelf life of the TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
When properly stored and handled, the TransIT-VirusGEN® Transfection Reagent is guaranteed for 1 year from the date of purchase. The TransIT-VirusGEN® GMP Transfection Reagent is guaranteed for a minimum of three months from the date of purchase with recertification of Retest Dates based on real-time stability data. If the Retest Date is updated it will be revised on the product Certificate of Analysis (CoA). Please contact techsupport@mirusbio.com for the CoA.

Q7. How should the TransIT-VirusGEN® Reagent be stored?
Store the TransIT-VirusGEN® Reagent from -10 to -30ºC. Before each use, warm to room temperature and vortex gently.

Q8. Can the TransIT-VirusGEN® Reagent be used to generate lentivirus and AAV in both adherent and suspension HEK 293 cells?
Yes. TransIT-VirusGEN® Reagent enables high-titer lentivirus and AAV production in both adherent, serum-containing cultures, as well as serum-free suspension 293-derived cell types. For optimal results, culture cells in the appropriate medium per cell type, with or without serum (e.g. DMEM + 10% FBS + 10 mM HEPES pH 7.4 for adherent 293T cultures; Expi293™ Expression Medium for suspension 293 cultures).

Q9. What titers are expected when generating virus with the TransIT-VirusGEN® Reagent?
Virus titers vary widely and ultimately depend on the HEK 293 cell subtype, packaging system and transfer vector used for lentivirus or AAV generation. For more information, see the TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent product pages.

PROTOCOL QUESTIONS AND ANSWERS

Q10. Which serum-free medium should I use for preparing TransIT-VirusGEN® Reagent:DNA transfection complexes?
For best results, we recommend using Phosphate Buffered Saline (PBS) without calcium or magnesium for preparing TransIT-VirusGEN® Reagent:DNA complexes. Serum-free media formulations such as Opti-MEM® I Reduced-Serum Medium can also be used. NOTE: Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes and should be avoided.

Q11. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Our starting recommendation is 15-60 min. However, users should empirically determine optimal complex formation time for each unique experimental system. Incubating complexes at 4°C may allow for longer, optimal complex formation times. Importantly, complexes should not be agitated after the initial mixing of reagent and DNA, which may disrupt complexes and result in decreased viral titers.

Q12. For lentivirus generation, how much TransIT-VirusGEN® Reagent and total DNA (packaging + transfer plasmid) should be used for transfection?
The quantities of TransIT-VirusGEN® Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel for adherent 293 cultures or the cell culture volume for suspension 293 cultures (see Table 1 and 2 in the TransIT-VirusGEN® Full Protocol (PDF)). In each T75 flask, our starting recommendation is 45 µl TransIT-VirusGEN® Reagent and 15 µg total DNA (7.5 µg transfer plasmid + 7.5 µg Packaging Mix) which represents a 3:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q13. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation?
The TransIT-VirusGEN® Reagent was optimized using a pre-mix of lentivirus packaging vectors. If using individual packaging plasmids, we recommend a starting ratio of 4 µg gag-pol vector, 1 µg rev vector and 1 µg VSV-G vector. Premix the packaging plasmids with an equal amount of the transfer vector (e.g. 6 µg) to maintain a 1:1 (wt:wt) ratio of packaging to transfer plasmids.

Q14. For AAV generation, how much TransIT-VirusGEN® Reagent and total DNA (GOI containing transfer + rep/cap + pHelper) should be used for transfection?
The quantities of TransIT-VirusGEN® Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel for adherent 293 cultures or the cell culture volume for suspension 293 cultures (see Table 3 and 4 in the TransIT-VirusGEN® Full Protocol (PDF)). In each T75 flask, our starting recommendation is 45 µl TransIT-VirusGEN® Reagent and 22.5 µg total DNA which represents a 2:1 ratio (µl of reagent/µg of DNA, vol:wt). For a 15 ml suspension culture, our starting recommendation is 45 µl TransIT-VirusGEN® Reagent and 30 µg total DNA which represents a 1.5:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q15. What is the recommended plasmid ratio for optimal AAV generation?
The TransIT-VirusGEN® Reagent was optimized using a 1:1:1 weight ratio of pAAV-hrGFP, pAAV-RC and pHelper (AAV Helper-Free System, Agilent Technologies).

Q16. How should transfections with TransIT-VirusGEN® Reagent be scaled if using culture vessels not listed in the Full Protocol?

  • For suspension 293 transfections, use the scaling charts for lentivirus and AAV Production in the TransIT-VirusGEN® Full Protocol (PDF) to scale per milliliter of total culture.
  • For adherent 293T culture transfections in alternate culture vessels, scale the amounts of TransIT-VirusGEN® Reagent, total DNA and serum-free medium for complex formation according to the surface area of the vessel.
    • For AAV transfection scaling: combine 0.02 ml PBS + 0.3 µg total DNA + 0.6 µl TransIT-VirusGEN® Reagent per cm2.
    • For lentivirus transfection scaling: combine + 0.02 ml PBS + 0.2 µg total DNA + 0.6 µl TransIT-VirusGEN® Reagent per cm2.

Q17. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q18. Is a media change required post-transfection?
No. A media change to remove TransIT-VirusGEN® Reagent:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus and AAV titers.

Q19. Are cell density and passage number important factors for successful transfection?
Yes. For adherent 293T cell types, a culture density of 80-95% confluence at the time of transfection is essential for optimal transfection using TransIT-VirusGEN® Reagent. Transfecting at less than 80% confluence may lead to increased cytotoxicity and decreased lentivirus and AAV titers. Monitoring passage number is important as high passage numbers can alter cell characteristics such as morphology and division rate. Maintain HEK 293T/17 cells below passage 30 for optimal recombinant lentivirus production. For suspension 293 cell types, the recommended cell density is 2 × 106 cells/ml. Passage cells 18-24 hours before transfection to ensure that cells are actively dividing and reach the appropriate density at time of transfection.

Q20. At what time point do you recommend harvesting lentivirus post-transfection?
The optimal incubation time for harvesting lentivirus is generally 48 hours post-transfection. Less lentivirus is produced during the period of 48-72 hours post-transfection.

Q21. How can I titer my lentivirus stock?
p24 ELISA assays designed to measure viral-associated p24 capsid protein and qPCR/ddPCR assays designed to amplify a conserved region of the HIV-1 genome (e.g. gag) are common methods for titering lentivirus. However, these methods measure the total amount of viral protein or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The TransIT-VirusGEN® Full Protocol (PDF) describes a method to determine functional titer by transducing HEK 293T/17 cells with a GFP reporter lentivirus. In this method, cells are transduced at a low MOI and assayed for GFP expression after 48 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (Transducing Units/ml) = (Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]) / (Volume of Lentivirus Stock in ml)

Q22. At what time point do you recommend harvesting AAV post-transfection?
The optimal incubation time for harvesting high-titer AAV is generally 48-72 hours post-transfection but should be determined empirically for each unique experimental system.

Q23. How can I titer my AAV stock?
ELISA assays designed to measure intact AAV particles and qPCR/ddPCR assays measuring the viral nucleic acid content of purified viruses (genome copy/ml) are commonly used to titer AAV. These methods measure particles or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The TransIT-VirusGEN® Full Protocol (PDF) describes a method to determine functional titer by transducing HT-1080 cells with a GFP reporter AAV. In this method, cells are transduced at a low MOI and assayed for GFP expression after 72 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (HT1080 Transducing Units/ml) = (Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]) / (Volume of AAV Stock in ml)

Reference Card: Methods to Measure Lentivirus and AAV in Your Sample (PDF)

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q24. How can I further improve titers when using TransIT-VirusGEN® Reagent?
Mirus Bio has developed Enhancer solutions which further boost AAV and lentivirus titers when used in conjunction with TransIT-VirusGEN® Reagent; the Enhancer solutions are sold together as the VirusGEN® AAV and VirusGEN® LV Transfection Kits. For more information about the VirusGEN® AAV Transfection Kit and VirusGEN® LV Transfection Kit, see the TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent product pages.

Multiple factors can contribute to lentiviral and AAV titers; refer to the following critical troubleshooting tips to improve your transfection results:

  • Optimize cell density (% confluence) at time of transfection - The recommended cell density for adherent 293T cell types at the time of transfection with TransIT-VirusGEN® Reagent is 80-95% confluence. For suspension 293 cells, the recommended density for transfection is 2×106 cells/ml.
  • Optimize TransIT-VirusGEN® Reagent to DNA ratio - For lentivirus generation, we recommend using 3 µl TransIT-VirusGEN® Reagent per 1 µg total DNA (transfer + packaging plasmids). For AAV generation, we recommend using 1.5-2 µl TransIT-VirusGEN® Reagent per 1 µg total DNA.
  • Use high quality DNA - DNA used for transfection should be highly purified, sterile and free from contaminants such as endotoxin. Remove any traces of endotoxin (e.g. bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Do NOT incubate TransIT-VirusGEN® Reagent without DNA - Diluting TransIT-VirusGEN® Reagent can be performed, but diluted reagent should be mixed with DNA within 5 minutes for optimal transfection complex formation. NOTE: TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components.
  • Do NOT disturb TransIT-VirusGEN® Reagent:DNA complexes after formation - After the initial mixing of TransIT-VirusGEN® Reagent with DNA, do not agitate or mix the complexes further. Agitation of the complexes will likely result in suboptimal functional virus production.
  • Form complexes in appropriate complex formation solution volume - We recommend forming transfection complexes in 10% of total culture volume, e.g. form complexes in 2.5 ml serum-free medium to transfect 25 ml culture volume. Increasing or decreasing the complex formation solution volume will require optimization of complex formation time.
  • Cells which are normally cultured in serum-containing medium should remain in serum-containing medium during and post-transfection - Though complex formation steps should occur in PBS or serum-free medium (e.g. Opti-MEM®), the cells themselves should be maintained in their normal complete (serum-containing) medium. A cell culture medium change is not necessary.
  • Optimize quantity of DNA used for transfection - Refer to Tables 1-4 in the TransIT-VirusGEN® Full Protocol (PDF) for recommended total DNA per reaction. Using amounts out of range of the recommended amounts of total DNA will likely result in suboptimal functional virus production.

For additional tips, please refer to the Troubleshooting Guide (PDF) in the user protocol.

Q25. Should I be concerned about cellular toxicity when using TransIT-VirusGEN® Reagent?
Morphology changes in HEK 293T cells following transfection with AAV plasmids are expected and indicate virus production. When over-expressing VSV-G in cultures (for lentivirus generation), some morphology changes and cell to cell fusion events are expected and do not adversely affect lentivirus titers. However, cellular toxicity that impacts viral titers may be observed if the transfection protocol is not followed properly. The following are considerations for improving cell health post-transfection:

  • Cell density (% confluence) too low at time of transfection - The recommended cell density at the time of transfection for adherent 293T cell types is 80-95% confluence. If cytotoxicity is observed, increase the cell density to maintain cell health.
  • Plasmid DNA contains high level of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired, consider reducing the amount of target plasmid. Maintain the optimal TransIT-VirusGEN® Reagent:DNA ratio by using carrier DNA such as an empty cloning vector. For example, use 0.5 µg transfer DNA, 0.5 µg pUC18 and 1 ug packaging plasmids for complexes with a reduced amount of target plasmid.
  • TransIT-VirusGEN® Reagent:DNA complexes were not mixed thoroughly with the cells in the well - Mix thoroughly to evenly distribute the complexes to all cells. Rock the dish back and forth and side to side to properly distribute the complexes. Do not swirl or rotate the dish, as this may result in uneven distribution. To ensure even complex distribution in larger culture vessels, sterilely remove the complete culture medium from cells and combine with transfection complexes (following the recommended complex incubation) and mix well before adding media + transfection complexes back to the vessel containing cells.
  • Cell morphology has changed - If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
  • Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a freshly thawed stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide (PDF) in the user protocol.

VirusGEN® AAV Transfection Kit

The VirusGEN® AAV Transfection Kit, available in Research Use Only (RUO) and GMP formats, enables high titer AAV production in suspension 293-derived cell types. The components of the VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit are identical in formulation, which equips researchers with the same reliable and scalable performance and streamlined viral vector production workflow as they progress from discovery and development to clinical trials and commercial manufacturing. The VirusGEN® GMP AAV Transfection Kit undergoes release testing for sterility, formulation identity, mycoplasma and endotoxin. Building on our commitment to product quality, the VirusGEN® GMP AAV Transfection Kit is manufactured under Current Good Manufacturing Practices (cGMP), which follow the regulations defined in U.S. 21 CFR 820 from the FDA.

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in the VirusGEN® AAV Transfection Kit?
Q2. What is the difference between VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit?
Q3. Is a functional AAV titer assay performed for quality release testing of the VirusGEN® GMP AAV Transfection Kit?
Q4. What analytical assays are available for VirusGEN® AAV Transfection Kit?
Q5. What configurations are available for VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit?
Q6. What is the composition of TransIT-VirusGEN® Reagent?
Q7. What is the composition of VirusGEN® AAV Complex Formation Solution and Enhancer?
Q8. What is the mechanism of action of VirusGEN® AAV Transfection Kit?
Q9. What is the shelf life of VirusGEN® AAV Transfection Kit?
Q10. How should VirusGEN® AAV Transfection Kit be stored?
Q11. Can VirusGEN® AAV Transfection Kit be used to generate AAV in both adherent and suspension cells?
Q12. What titers are expected when using VirusGEN® AAV Transfection Kit?

PROTOCOL QUESTIONS AND ANSWERS

Q13. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
Q14. Can I filter TransIT-VirusGEN® Reagent?
Q15. For AAV generation, how much TransIT-VirusGEN® Reagent, total DNA (GOI containing transfer + rep/cap + pHelper) and VirusGEN® Complex Formation Solution and Enhancer should be used for transfection?
Q16. What is the recommended plasmid ratio for optimal AAV generation?
Q17. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Q18. Will antibiotics interfere with my transfection?
Q19. Is a media change required post-transfection?
Q20. Are cell density and passage number important factors for successful transfection?
Q21. At what time point do you recommend harvesting AAV post-transfection?
Q22. How can I titer my AAV stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. What can I do to further improve my AAV titers when using VirusGEN® AAV Transfection Kit?
Q24. Should I be concerned about cellular toxicity when using VirusGEN® AAV Transfection Kit?

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in VirusGEN® AAV Transfection Kit?
The VirusGEN® AAV Transfection Kit consists of two components: TransIT-VirusGEN® Transfection Reagent and VirusGEN® AAV Complex Formation Solution and Enhancer.

Q2. What is the difference between VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit?
The components of the three products have the same formulation, but each product has different quality control release requirements:

  • The TransIT-VirusGEN® Transfection Reagent is a research-grade product which is released based on appearance and a functional AAV titer assay for all volume configurations; additional release testing (sterility, endotoxin, and mycoplasma) is performed for configurations that are 1.5 ml or greater in volume. The 100-ml VirusGEN® AAV Complex Formation Solution and Enhancer is a research-grade product which is released based on appearance, sterility, identity, endotoxin, osmolality and pH. The 1-L VirusGEN® AAV Complex Formation Solution and Enhancer is a research-grade product which is released based on appearance, sterility, identity, mycoplasma, endotoxin, osmolality and pH.
  • The TransIT-VirusGEN® GMP Transfection Reagent and VirusGEN® GMP AAV Complex Formation Solution and Enhancer are manufactured in accordance with the Mirus Bio Quality Management System, which incorporates the appropriate cGMP product and production requirements. The products are manufactured in audited facilities that are certified to ISO 13485 and comply with 21 CFR 820. Release testing for TransIT-VirusGEN® GMP Transfection Reagent consists of appearance, sterility, identity, mycoplasma and endotoxin assays. In addition to these tests, the VirusGEN® GMP AAV Complex Formation Solution and Enhancer is assayed for pH and osmolality.

Q3. Is a functional AAV titer assay performed for quality release testing of the VirusGEN® GMP AAV Transfection Kit?
A functional AAV titer assay is not performed for quality release testing of the VirusGEN® GMP AAV Transfection Kit. The VirusGEN® GMP AAV Transfection Kit is formulated through a validated manufacturing process which was established to ensure consistent functional AAV titer thereby removing the need to perform a functional AAV titer assay on each lot.

Q4. What analytical assays are available for VirusGEN® AAV Transfection Kit?
Residual reagent and identity tests are available for VirusGEN® products. Please contact techsupport@mirusbio.com for more information.

Q5. What configurations are available for VirusGEN® AAV Transfection Kit and VirusGEN® GMP AAV Transfection Kit?

  • VirusGEN® AAV Transfection Kit is available in three formats (MIR 6750, MIR 6745 and MIR 6755). MIR 6750 includes 2×1.5 ml TransIT-VirusGEN® Transfection Reagent and 1×100 ml VirusGEN® AAV Complex Formation Solution and Enhancer. MIR 6745 includes 1×30 ml TransIT-VirusGEN® Transfection Reagent and 1×1 L VirusGEN® AAV Complex Formation Solution and Enhancer. MIR 6755 includes 1×150 ml TransIT-VirusGEN® Transfection Reagent and 5×1 L VirusGEN® AAV Complex Formation Solution and Enhancer.
  • VirusGEN® GMP AAV Transfection Kit (MIR 6815-GMP) includes 1×150 ml TransIT-VirusGEN® GMP Transfection Reagent and 5×1 L VirusGEN® GMP AAV Complex Formation Solution and Enhancer.

Q6. What is the composition of TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent is a proprietary, animal origin free formulation composed of a lipid and polymer mixture.

Q7. What is the composition of VirusGEN® AAV Complex Formation Solution and Enhancer?
VirusGEN® AAV Complex Formation Solution and Enhancer is a proprietary, chemically defined and animal origin free formulation.

Q8. What is the mechanism of action of each of the components of VirusGEN® AAV Transfection Kit?
TransIT-VirusGEN® Reagent complexes with negatively charged DNA to form lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are taken into the cell via endocytosis. We believe the enhancer component increases production of certain viral proteins when added between 0-24 hours post-transfection. The enhancer component does not directly impact transfection efficiency because improved AAV titers are observed even when the enhancer component is added several hours post-transfection. 

Q9. What is the shelf life of VirusGEN® AAV Transfection Kit?
For the VirusGEN® AAV Transfection Kit: the TransIT-VirusGEN® Transfection Reagent component is guaranteed for 1 year from the date of purchase, and the VirusGEN® AAV Complex Formation Solution and Enhancer is guaranteed for 6 months from the date of purchase. All components of the VirusGEN® GMP AAV Transfection Kit are guaranteed for a minimum of three months from the date of purchase with recertification of Retest Dates based on real-time stability data. If the Retest Date is updated it will be revised on the product Certificate of Analysis (CoA). Please contact techsupport@mirusbio.com for the CoA.

Q10. How should VirusGEN® AAV Transfection Kit be stored?
Store TransIT-VirusGEN® Reagent from -10 to -30°C. Store VirusGEN® AAV Complex Formation Solution and Enhancer from 4 to 10°C. 

Q11. Can VirusGEN® AAV Transfection Kit be used to generate AAV in both adherent and suspension cells?
VirusGEN® AAV Complex Formation Solution and Enhancer is only recommended for suspension HEK 293 cell lines. Generally, use of VirusGEN® AAV Complex Formation Solution and Enhancer in adherent HEK 293 cell lines is ineffective or even detrimental to AAV production. However, TransIT-VirusGEN® Reagent component is compatible for high titer AAV production in both adherent and suspension 293-derived cell types and can be purchased separately from the enhancer component of VirusGEN® AAV Transfection Kit.

Q12. What titers are expected when using VirusGEN® AAV Transfection Kit?
Virus titers vary widely and ultimately depend on the HEK 293 cell subtype, packaging system and transfer vector and serotype used for AAV generation. For more information, see VirusGEN® AAV Transfection Kit.

PROTOCOL QUESTIONS AND ANSWERS

Q13. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
For best results, we recommend using AAV Complex Formation Solution and Enhancer for preparing TransIT-VirusGEN® Reagent:DNA complexes. If you would like to evaluate transfection without the enhancer component (i.e. without AAV Complex Formation Solution and Enhancer), we recommend using Phosphate Buffered Saline (PBS) without calcium or magnesium. Serum-free media formulations such as Opti-MEM® I Reduced-Serum Medium can also be used. NOTE: Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes and should be avoided.

Q14. Can I filter TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent can be filtered with no effect on reagent performance if it has not been diluted in aqueous solution. TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components.

Q15. For AAV generation, how much TransIT-VirusGEN® Reagent, total DNA (GOI containing transfer + rep/cap + pHelper) and VirusGEN® Complex Formation Solution and Enhancer should be used for transfection? 
The quantities of TransIT-VirusGEN® Reagent, total DNA and Complex Formation Solution and Enhancer needed for transfection will depend upon cell culture vessel and volume (see Table 1 in the VirusGEN® AAV Transfection Kit Full Protocol). Per 1 ml of complete growth medium, our starting recommendation is 3 µl TransIT-VirusGEN® Reagent and 2 µg total DNA in 100 µl of VirusGEN® AAV Complex Formation Solution and Enhancer, which represents a 1.5:1 ratio (µl of reagent/µg of DNA, vol:wt) in 10% culture volume.

Q16. What is the recommended plasmid ratio for optimal AAV generation?
The TransIT-VirusGEN® Reagent was optimized using a 1:1:1 weight ratio of pAAV-hrGFP, pAAV-RC and pHelper (AAV Helper-Free System, Agilent Technologies). The optimal ratio may differ by serotype and packaging system and should be empirically determined for each unique experimental system.

Q17. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Our starting recommendation is 15-60 min. However, users should empirically determine optimal complex formation time for each unique experimental system. Incubating complexes at 4°C may allow for longer, optimal complex formation times.  Importantly, complexes should not be agitated after the initial mixing of reagent and DNA, which may disrupt complexes and result in decreased viral titers.

Q18. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q19. Is a media change required post-transfection?
No. A media change to remove TransIT-VirusGEN® Reagent:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to AAV titers.

Q20. Are cell density and passage number important factors for successful transfection?
Yes. For suspension 293 cell types, the recommended cell density at transfection is 2×106 cells/ml. Passage cells 18-24 hours before transfection to ensure that cells are actively dividing and reach the appropriate density at time of transfection.

Q21. At what time point do you recommend harvesting AAV post-transfection?
The optimal incubation time for harvesting high-titer AAV is generally 48-72 hours post-transfection but should be determined empirically for each unique experimental system

Q22. How can I titer my AAV stock?
ELISA assays designed to measure intact AAV particles and qPCR/ddPCR assays measuring the viral nucleic acid content of purified viruses (genome copy/ml) are commonly used to titer AAV. These methods measure particles or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The VirusGEN® AAV Transfection Kit Full Protocol describes a method to determine functional titer by transducing HT-1080 cells with a GFP reporter AAV2. In this method, cells are transduced at a low MOI and assayed for GFP expression after 72 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (HT-1080 Transducing Units/ml) = Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]
(Volume of AAV2 Stock in ml)

Reference Card: Methods to Measure Lentivirus and AAV in Your Sample (PDF)

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. What can I do to further improve my AAV titers when using VirusGEN® AAV Transfection Kit?
Multiple factors can contribute to AAV titers; refer to the following critical troubleshooting tips to improve your transfection results:

  • Optimize cell density at time of transfection - The recommended cell density for suspension 293 cells at the time of transfection is 2×106 cells/ml.  
  • Optimize TransIT-VirusGEN® Reagent to DNA ratio - For AAV generation, we recommend using 3 µl TransIT-VirusGEN® Reagent per 2 µg total DNA.
  • Use high quality DNA - DNA used for transfection should be highly purified, sterile and free from contaminants such as endotoxin. Remove any traces of endotoxin (e.g. bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Do NOT incubate TransIT-VirusGEN® Reagent without DNA - Diluting TransIT-VirusGEN® Reagent can be performed, but diluted reagent should be mixed with DNA within 5 minutes for optimal transfection complex formation. NOTE: TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components. 
  • Do NOT disturb TransIT-VirusGEN® Reagent:DNA complexes after formation - After the initial mixing of TransIT-VirusGEN® Reagent with DNA, do not agitate or mix the complexes further. Agitation of the complexes will likely result in suboptimal functional virus production.
  • Form complexes in appropriate complex formation solution volume – We recommend forming transfection complexes in 10% of total culture volume, e.g. form complexes in 2.5 ml VirusGEN® AAV Complex Formation Solution and Enhancer (or PBS) to transfect 25 ml culture volume. Increasing or decreasing the complex formation solution volume will require optimization of complex formation time.
  • Cells which are normally cultured in serum-containing medium should remain in serum-containing medium during and post-transfection – Though complex formation steps should occur in serum-free medium (e.g. VirusGEN® AAV Complex Formation Solution and Enhancer or PBS), the cells themselves should be maintained in their normal complete (serum-containing) medium. A cell culture medium change is not necessary.
  • Optimize quantity of DNA used for transfection - Refer to Table 1 in the VirusGEN® AAV Transfection Kit Full Protocol for recommended total DNA per reaction. Using amounts out of range of the recommended amounts of total DNA will likely result in suboptimal functional virus production.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Q24. Should I be concerned about cellular toxicity when using VirusGEN® AAV Transfection Kit?
When generating AAV with VirusGEN® AAV Complex Formation Solution and Enhancer, cell growth may decrease. This is normal and does not adversely affect virus titers. However, cellular toxicity that impacts viral titers may be observed if the transfection protocol is not followed properly. The following are considerations for improving cell health post-transfection:

  • Cell density too low at time of transfection - If cytotoxicity is observed, increase the cell density to maintain cell health.
  • Plasmid DNA contains high level of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired, consider reducing the amount of target plasmid. Maintain the optimal TransIT-VirusGEN® Reagent:DNA ratio by using carrier DNA such as an empty cloning vector. For example, use 0.5 µg transfer DNA, 0.5 µg pUC18 and 1 ug packaging plasmids for complexes with a reduced amount of target plasmid.
  • Cell morphology has changed – If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
  • Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a freshly thawed stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

VirusGEN® LV Transfection Kit

The VirusGEN® LV Transfection Kit, available in Research Use Only (RUO) and GMP formats, enables high titer lentivirus production in adherent and suspension 293-derived cell types. The components of the VirusGEN® LV Transfection Kit and VirusGEN® GMP LV Transfection Kit are identical in formulation, which equips researchers with the same reliable and scalable performance and streamlined viral vector production workflow as they progress from discovery and development to clinical trials and commercial manufacturing. The VirusGEN® GMP LV Transfection Kit undergoes release testing for sterility, formulation identity, mycoplasma and endotoxin. Building on our commitment to product quality, the VirusGEN® GMP LV Transfection Kit is manufactured under Current Good Manufacturing Practices (cGMP), which follow the regulations defined in U.S. 21 CFR 820 from the FDA.

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in VirusGEN® LV Transfection Kit?
Q2. What is the difference between VirusGEN® LV Transfection Kit and VirusGEN® GMP LV Transfection Kit?
Q3. Is a functional AAV titer assay performed for quality release testing of TransIT-VirusGEN® GMP Transfection Reagent?
Q4. What analytical assays are available for VirusGEN® LV Transfection Kit?
Q5. What configurations are available for VirusGEN® LV Transfection Kit and VirusGEN® GMP LV Transfection Kit?
Q6. What is the composition of TransIT-VirusGEN® Reagent?
Q7. What is the composition of VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer?
Q8. What is the mechanism of action of each of the components of VirusGEN® LV Transfection Kit?
Q9. What is the shelf life of VirusGEN® LV Transfection Kit?
Q10. How should VirusGEN® LV Transfection Kit be stored?
Q11. Can VirusGEN® LV Transfection Kit be used to generate lentivirus in both adherent and suspension cells?
Q12. What titers are expected when using VirusGEN® LV Transfection Kit?

PROTOCOL QUESTIONS AND ANSWERS

Q13. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
Q14. Can I filter TransIT-VirusGEN® Reagent?
Q15. How much TransIT-VirusGEN® Reagent and total DNA (transfer + packaging plasmids) should be used for transfection?
Q16. What is the recommended transfer:packaging plasmid ratio for optimal lentivirus generation?
Q17. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Q18. When should VirusGEN® LV Enhancer be added to the culture?
Q19. Will antibiotics interfere with my transfection?
Q20. Is a media change required post-transfection?
Q21. Are cell density and passage number important factors for successful transfection?
Q22. At what time point do you recommend harvesting lentivirus post-transfection?
Q23. How can I titer my lentivirus stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q24. What can I do to further improve my lentivirus titers when using VirusGEN® LV Transfection Kit?
Q25. Should I be concerned about cellular toxicity when using VirusGEN® LV Transfection Kit?

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in VirusGEN® LV Transfection Kit?
The VirusGEN® LV Transfection Kit consists of three components: TransIT-VirusGEN® Transfection Reagent, VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer.

Q2. What is the difference between VirusGEN® LV Transfection Kit and VirusGEN® GMP LV Transfection Kit?
The components of the three products have the same formulation, but each product has different quality control release requirements:

  • The TransIT-VirusGEN® Transfection Reagent is a research-grade product which is released based on appearance and a functional AAV titer assay for all volume configurations; additional release testing (sterility, endotoxin, and mycoplasma) is performed for configurations that are 1.5 ml or greater in volume. The 100-ml size VirusGEN® LV Complex Formation Solution is a research-grade product which is released based on appearance, sterility, endotoxin, osmolality and pH. The 100-ml size VirusGEN® LV Enhancer is a research-grade product which is released based on appearance, sterility, identity, endotoxin, osmolality and pH. The 1-L size VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer are research-grade products which are released based on sterility, identity, mycoplasma, endotoxin, osmolality and pH.
  • The TransIT-VirusGEN® GMP Transfection Reagent, VirusGEN® GMP LV Complex Formation Solution and VirusGEN® GMP LV Enhancer are manufactured in accordance with the Mirus Bio Quality Management System, which incorporates the appropriate cGMP product and production requirements. The products are manufactured in audited facilities that are certified to ISO 13485 and comply with 21 CFR 820. Release testing for TransIT-VirusGEN® GMP Transfection Reagent consists of appearance, sterility, identity, mycoplasma and endotoxin assays. In addition to these tests, the VirusGEN® GMP LV Complex Formation Solution and VirusGEN® GMP LV Enhancer are assayed for pH and osmolality.

Q3. Is a functional viral titer assay performed for quality release testing of the VirusGEN® GMP LV Transfection Kit?
A functional viral titer assay is not performed for quality release testing of the VirusGEN® GMP LV Transfection Kit. The VirusGEN® GMP LV Transfection Kit is formulated through a validated manufacturing process which was established to ensure consistent functional viral titer thereby removing the need to perform a functional viral titer assay on each lot. 

Q4. What analytical assays are available for VirusGEN® LV Transfection Kit?
Residual reagent and identity tests are available for VirusGEN® products. Please contact techsupport@mirusbio.com for more information.

Q5. What configurations are available for VirusGEN® LV Transfection Kit and VirusGEN® GMP LV Transfection Kit?

  • VirusGEN® LV Transfection Kit is available in three formats (MIR 6760, MIR 6765, MIR 6792). MIR 6760 includes 2×1.5 ml TransIT-VirusGEN® Transfection Reagent, 1×100 ml VirusGEN® LV Complex Formation Solution and 1×100 ml VirusGEN® LV Enhancer. MIR 6765 includes 1×30 ml TransIT-VirusGEN® Transfection Reagent, 1×1 L VirusGEN® LV Complex Formation Solution and 1×1 L VirusGEN® LV Enhancer. MIR 6792 includes 1×150 ml TransIT-VirusGEN® Transfection Reagent, 5×1 L VirusGEN® LV Complex Formation Solution and 5×1 L VirusGEN® LV Enhancer.
  • VirusGEN® GMP LV Transfection Kit (MIR 6825-GMP) includes 1×150 ml TransIT-VirusGEN® GMP Transfection Reagent, 5×1 L VirusGEN® GMP LV Complex Formation Solution and 5×1 L VirusGEN® LV Enhancer.

Q6. What is the composition of TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent is a proprietary, animal origin free formulation composed of a lipid and polymer mixture.

Q7. What is the composition of VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer?
VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer are proprietary, chemically defined and animal origin free formulations.

Q8. What is the mechanism of action of each of the components of VirusGEN® LV Transfection Kit?
TransIT-VirusGEN® Reagent complexes with negatively charged DNA to form lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are taken into the cell via endocytosis. We believe the enhancer component increases production of certain viral proteins when added between 18-24 hours post-transfection. The enhancer component does not directly impact transfection efficiency because improved lentiviral titers are observed even when the enhancer component is added several hours post-transfection. 

Q9. What is the shelf life of VirusGEN® LV Transfection Kit?
For the VirusGEN® LV Transfection Kit: the TransIT-VirusGEN® Transfection Reagent component is guaranteed for 1 year from the date of purchase, and the VirusGEN® LV Complex Formation Solution and LV Enhancer are guaranteed for 6 months from the date of purchase. All components of the VirusGEN® GMP LV Transfection Kits are guaranteed for a minimum of three months from the date of purchase with recertification of Retest Dates based on real-time stability data. If the Retest Date is updated it will be revised on the product Certificate of Analysis (CoA). Please contact techsupport@mirusbio.com for the CoA.

Q10. How should VirusGEN® LV Transfection Kit be stored?
Store TransIT-VirusGEN® Reagent from -10 to -30°C. Store VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer from 4 to 10°C. 

Q11. Can VirusGEN® LV Transfection Kit be used to generate lentivirus in both adherent and suspension cells?
Yes. VirusGEN® LV Transfection Kit enables high titer lentivirus production in both adherent, serum-containing cultures, as well as serum-free suspension 293-derived cell types. For optimal results, culture cells in the appropriate medium per cell type, with or without serum (e.g. DMEM + 10% FBS + 10 mM HEPES pH 7.4 for adherent 293T cultures; FreeStyle™ F17 + 4 mM L-Glutamine + 0.2% Poloxamer 188 for suspension 293 cultures).

Q12. What titers are expected when using VirusGEN® LV Transfection Kit?
Virus titers vary widely and ultimately depend on the HEK 293 cell subtype, packaging system and gene on the transfer plasmid used for lentivirus generation. For more information, see VirusGEN® LV Transfection Kit.

PROTOCOL QUESTIONS AND ANSWERS

Q13. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
For best results, we recommend using VirusGEN® LV Complex Formation Solution for preparing TransIT-VirusGEN® Reagent:DNA complexes. Phosphate Buffered Saline (PBS) without calcium or magnesium or serum-free media formulations such as Opti-MEM® I Reduced-Serum Medium can also be used. NOTE: Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes and should be avoided.

Q14. Can I filter TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent can be filtered with no effect on reagent performance if it has not been diluted in aqueous solution. TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components.

Q15. How much TransIT-VirusGEN® Reagent and total DNA (transfer + packaging plasmids) should be used for transfection? 
The quantities of TransIT-VirusGEN® Reagent, total DNA, VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer needed for transfection will depend upon cell culture vessel and volume (see Table 1 and 2 in the VirusGEN® LV Transfection Kit Full Protocol). In each T75 flask, our starting recommendation is 45 µl TransIT-VirusGEN® Reagent and 15 µg total DNA (7.5 µg transfer plasmid + 7.5 µg Packaging Mix) which represents a 3:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q16. What is the recommended transfer:packaging plasmid ratio for optimal lentivirus generation?
The TransIT-VirusGEN® Reagent was optimized using a pre-mix of lentivirus packaging vectors. If using individual packaging plasmids, we recommend a starting ratio of 4 µg gag-pol vector, 1 µg rev vector and 1 µg VSV-G vector. Premix the packaging plasmids with an equal amount of the transfer vector (e.g. 6 µg) to maintain a 1:1 (wt:wt) ratio of packaging to transfer plasmids.

Q17. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Our starting recommendation is 15-60 min. However, users should empirically determine optimal complex formation time for each unique experimental system. Incubating complexes at 4°C may allow for longer, optimal complex formation times.  Importantly, complexes should not be agitated after the initial mixing of reagent and DNA, which may disrupt complexes and result in decreased viral titers.

Q18. When should VirusGEN® LV Enhancer be added to the culture?
VirusGEN® LV Enhancer should be added 18-24 hr post-transfection.

Q19. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q20. Is a media change required post-transfection?
No. A media change to remove TransIT-VirusGEN® Reagent:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus titers.

Q21. Are cell density and passage number important factors for successful transfection?
Yes. For adherent 293T cell types, a culture density of 80-95% confluence at the time of transfection is essential for optimal transfection using TransIT-VirusGEN® Reagent. Transfecting at less than 80% confluence may lead to increased cytotoxicity and decreased lentivirus titers. Monitoring passage number is important as high passage numbers can alter cell characteristics such as morphology and division rate. Maintain HEK 293T/17 cells below passage 30 for optimal recombinant lentivirus production. For suspension 293 cell types, the recommended cell density is 2 × 106 cells/ml. Passage cells 18-24 hours before transfection to ensure that cells are actively dividing and reach the appropriate density at time of transfection

Q22. At what time point do you recommend harvesting lentivirus post-transfection?
The optimal incubation time for harvesting lentivirus is generally 48 hours post-transfection. Less lentivirus is produced during the period of 48-72 hours post-transfection.

Q23. How can I titer my lentivirus stock?
p24 ELISA assays designed to measure viral-associated p24 capsid protein and qPCR/ddPCR assays designed to amplify a conserved region of the HIV-1 genome (e.g. gag) are common methods for titering lentivirus. However, these methods measure the total amount of viral protein or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The VirusGEN® LV Transfection Kit Full Protocol describes a method to determine functional titer by transducing HEK 293T/17 cells with a GFP reporter lentivirus. In this method, cells are transduced at a low MOI and assayed for GFP expression after 48 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (Transducing Units/ml) = Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]
(Volume of Lentivirus Stock in ml)

Reference Card: Methods to Measure Lentivirus and AAV in Your Sample (PDF)

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q24. What can I do to further improve my lentivirus titers when using VirusGEN® LV Transfection Kit?
Multiple factors can contribute to lentivirus titers; refer to the following critical troubleshooting tips to improve your transfection results:

  • Optimize cell density (% confluence) at time of transfection - The recommended confluency at the time of transfection for adherent 293T cell types is 80-95% confluence. For suspension 293 cells, the recommended density for transfection is 2×106 cells/ml.  
  • Optimize TransIT-VirusGEN® Reagent to DNA ratio - For lentivirus generation, we recommend using 3 µl TransIT-VirusGEN® Reagent per 1 µg total DNA (transfer + packaging plasmids).
  • Use high quality DNA - DNA used for transfection should be highly purified, sterile and free from contaminants such as endotoxin. Remove any traces of endotoxin (e.g. bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Do NOT incubate TransIT-VirusGEN® Reagent without DNA - Diluting TransIT-VirusGEN® Reagent can be performed, but diluted reagent should be mixed with DNA within 5 minutes for optimal transfection complex formation. NOTE: TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components. 
  • Do NOT disturb TransIT-VirusGEN® Reagent:DNA complexes after formation - After the initial mixing of TransIT-VirusGEN® Reagent with DNA, do not agitate or mix the complexes further. Agitation of the complexes will likely result in suboptimal functional virus production.
  • Form complexes in appropriate complex formation solution volume – We recommend forming transfection complexes in 10% of total culture volume, e.g. form complexes in 2.5 ml VirusGEN® LV Complex Formation Solution (or PBS) to transfect 25 ml culture volume. Increasing or decreasing the complex formation solution volume will require optimization of complex formation time.
  • Cells which are normally cultured in serum-containing medium should remain in serum-containing medium during and post-transfection - Though complex formation steps should occur in serum-free medium (e.g. VirusGEN® LV Complex Formation Solution or PBS), the cells themselves should be maintained in their normal complete (serum-containing) medium. A cell culture medium change is not necessary.
  • Optimize quantity of DNA used for transfection - Refer to Table 1 and 2 in the VirusGEN® LV Transfection Kit Full Protocol for recommended total DNA per reaction. Using amounts out of range of the recommended amounts of total DNA will likely result in suboptimal functional virus production.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Q25. Should I be concerned about cellular toxicity when using VirusGEN® LV Transfection Kit?
When generating lentivirus, overexpression of the vesicular stomatitis virus (VSV) G protein causes changes in cell morphology and can even result in cell-cell fusion. VirusGEN® LV Enhancer may also decrease cell growth. This is normal and does not adversely affect virus titers. However, cellular toxicity that impacts viral titers may be observed if the transfection protocol is not followed properly. The following are considerations for improving cell health post-transfection:

  • Cell density (% confluence) too low at time of transfection - The recommended confluency at the time of transfection for adherent 293T cell types is 80-95% confluence. For suspension 293 cells, the recommended density for transfection is 2×106 cells/ml. If cytotoxicity is observed, increase the cell density to maintain cell health.
  • Plasmid DNA contains high level of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
  • Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired, consider reducing the amount of target plasmid. Maintain the optimal TransIT-VirusGEN® Reagent:DNA ratio by using carrier DNA such as an empty cloning vector. For example, use 0.5 µg transfer DNA, 0.5 µg pUC18 and 1 ug packaging plasmids for complexes with a reduced amount of target plasmid.
  • TransIT-VirusGEN® Reagent:DNA complexes were not mixed thoroughly with the cells in the well - Mix thoroughly to evenly distribute the complexes to all cells. Rock the dish back and forth and side to side to properly distribute the complexes. Do not swirl or rotate dishes, as this may result in uneven distribution. To ensure even complex distribution in larger culture vessels, sterilely remove the complete culture medium from cells and combine with transfection complexes (following the recommended complex incubation) and mix well before adding media + transfection complexes back to the vessel containing cells
  • Cell morphology has changed – If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
  • Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a freshly thawed stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

 


Technical Resources

Don’t See Your Cell Type? Consult Reagent Agent® Transfection Database
Citation Database: Check if our reagents have been used by other researchers to transfect your cell type
Technical Support: Communicate directly with a transfection expert

"We recently engineered a bispecific immunofusion for the treatment and elimination of leukemia stem cells. For this work we chose TransIT-PRO® for antibody production in CHO-S cells based on the high protein yield we obtained. (Kuo et al. Protein Eng Des Sel. 2012 Oct;25(10):561-9. Epub 2012 Jun 27)."
Jen-Sing Liu, Ph.D.
Molecular Templates Inc.